Bacteria naturally associated with the host (commensals) limit disease caused by invading pathogens; however, the mechanisms utilized by beneficial microbes to inhibit pathogenesis are poorly understood. To elucidate the role of commensals in intestinal health, we use an animal model in which mice are colonized with the commensal microbe Bacillus subtilis prior to infection with the attaching and effacing (A/E) pathogen, Citrobacter rodentium. Gram positive spore-forming B. subtilis is present in mammalian gastrointestinal tracts and is safely consumed by humans. C. rodentium is genetically and functionally similar to the human A/E pathogens, enteropathogenic E. coli and enterohemorrhagic E. coli. Mice colonized with commensal wt B. subtilis prior to C. rodentium infection exhibited less diarrheal and intestinal disease than mice without B. subtilis. Further, we discovered that B. subtilis mutants unable to synthesize either an exopolysaccharide or flagellin do not protect the host from C. rodentium-associated disease. Bacterial exopolysaccharides and flagellin are ligands for the host receptors, TLR2 and TLR5, respectively. Since B. subtilis mutants deficient in these TLR ligands do not protect mice from C. rodentium-associated disease, we hypothesize that TLR signaling is required for B. subtilis-mediated protection. The innate immune response includes B1 lymphocytes which produce low affinity polyreactive natural antibodies. Natural antibodies can bind enteric pathogens and limit disease. Commensal colonization promotes B1 lymphocyte migration into the intestinal lamina propria. Commensal B. subtilis could promote the migration of natural antibody-producing B1 cells to the intestine and natural antibodies may limit intestinal inflammation by preventing C. rodentium from attaching to the gut epithelium. We hypothesize that natural antibody-producing B1 cells are required for B. subtilis-mediated protection from C. rodentium-associated disease. The goal of these studies is to determine if commensals require TLR signaling and host B1 lymphocytes to protect the host from C. rodentium-associated disease. To test these ideas, we will orally gavage specific-pathogen-free C57BL/6, TLR2 KO, TLR5 KO, and B1 lymphocyte depleted mice with wt B. subtilis and determine if susceptibility to C. rodentium infection is altered. We will also determine if B. subtilis colonization promotes migration of B1 cells into the intestinal lamina propria. Results from these studies will elucidate important commensal:host interactions that limit intestinal inflammation. Host:commensal interactions are critical for immune homeostasis and for limiting disease caused by invading pathogens. By improving our current knowledge of how commensals and the host cooperate to prevent inflammation, we could better utilize commensals to prevent disease induced by pathogenic bacteria.